Keywords

Introduction

One of the pressing environmental problems facing our country today is the effluents from mining industries generating large volume of wastewater contaminated with heavy metals that pollute ground water, deplete aquatic life, which contaminate the food chain and deteriorate the ecosystem. Water discharge from mine tailings is usually acidic in nature and contains high concentration of heavy metals ions. The presence of heavy metals in aquatic environment is known to cause severe damage to aquatic life, and could eliminate the microorganisms needed for biological treatment of wastewater and consequently delay the process of water purification.

The study aims to apply microbial isolates for the biological treatment of acidic wastewater and reduction of heavy metals and to develop a cost-effective treatment system that would be able to remediate abandoned mine sites. In particular, a biological treatment system will be designed and operated employing microorganisms found on site. This will involve the screening, isolation and purification of best pollutant degrading bacteria found in Itogon, Benguet abandoned mine site. The preselected microorganisms will be tested for their removal efficiencies both on a batch and continuous basis. Physico-chemical methods such as: chemical precipitation, oxidation, and reduction, electrochemical treatment, evaporative recovery, filtration, ion exchange and membrane technologies have been widely used to remove heavy metal ions from industrial wastewater others (Das et al. 2008). These processes may be ineffective or expensive (Liu et al., 2003; Pamukoglu & Kargi, 2006). On the other hand, biological treatment is considered a promising technique for bioremediation of heavy metals wastewater, since it can degrade organic pollutant in the wastewater and simultaneously transform heavy metals (Wang et al. 2010). Biological treatments constitute an alternative method for the removal of heavy metals. Through his process we eliminate pollution in the area and restore aquatic life.

Materials and Methods

All reagents were analytical grade. Different concentrations of heavy metals such as: Copper Sulphate (CuSO4.5H2O) and Zinc Sulphate (ZnSO4.7H2O) were obtained from Univar. Potassium Dichromate (K2Cr2O7) were from Qualikems made in India, Lead Nitrate (Pb(NO3)2 and Nickel Sulphate (Ni2(SO4)) from Merck. Nutrient Agar (NA) and Nutrient Broth (NB) were from HiMedia made in India. Plastic cups were obtained from recycled bottles.

Wastewater Sampling

The water samples were collected from six (6) different locations in mine tailings at Itogon, Benguet, Mt. Province. The sampling sites are as follows:

Site

Label

Location (Barangay) in Itogon Benguet

1

Balatoc

Virac

2

Ambalanga River, Phase 3, Pink Tunnel, Narba Tunnel

Ucab

3

TSF 1 ( Gold Creek)

Ucab

4

Convergence Ambalanga, Acupan River

Poblacion

5

TSF 2 ( Active)

Poblacion

6

Antamok 440

Loacan

Three (3) samples were collected from the center of the river and the 3 samples from the riverbanks. The collected water from each location was poured into a single sample bottle (1 Liter each) to a total of six bottles. Proper coding and identification of the sampling bottle with corresponding pH was labelled. The samples were stored in an ice cooler box and transported to the laboratory for isolation of heavy metal resistant microorganisms.

Isolation of Organisms from Contaminated Wastewater

Microbial samples were sourced from the contaminated wastewater from various locations at the mining sites in Benguet Province. One ml each from wastewater samples were diluted in 9 ml sterile water. Serial dilution up to 10-5 and a 0.1 ml diluted wastewater was then spread to the nutrient agar plates with different concentrations of heavy metals (5, 10, 15, 20 ppm each). The plates were then incubated at room temperature for 24 hours. Then picked and streaked the best selected isolates in NA plates with mixed heavy metals (60 and 100 ppm) concentrations, then incubated at room temperature for 24 hours. Select best isolates for growth at high concentration 100 ppm of heavy metal and streaked on NA test tubes slants, incubated at room temperature for 24 hours and stored in refrigerator for screening.

Screening of Microorganisms and Sample Digestion with HCl

From 150 initial isolates, fifty (50) selected strains were screened in NA plates with different concentrations of heavy metals (i.e., 5, 10, 15, 20, 60 and 100 ppm). Then 4 isolates were grown in 100 ppm concentration of heavy metals Cr, Zn, Ni, and Pb. The resulting 4 strains were cultured in flasks using various concentrations of heavy metals 25, 50, 75 and 100 ppm and nutrient broth. Instead of distilled water, synthetic mines water (SMW) containing (CuSO4.5H2O, ZnSO4.5H2O, K2Cr2O7, Pb(NO3)2, Ni2(SO4) was used as diluent. Then a 100 ml of synthetic mines water and nutrient broth was cultured with 10% inoculant of a selected isolate. After 5 days in a shaker at room temperature the samples were filtered and digested with concentrated HCl and read at the atomic absorption spectrophotometer (PinAAcle 500 AAS, Perkin Elmer, USA).

Semi Continuous Biosorption Experiment

Inoculum propagation for both screening and for biosorption of heavy metals was done in nutrient broth media. Nutrient agar slants were streaked with stock culture of each chosen organism (Acinetobacter sp. junii) and incubated for 24 hours at room temperature. The organism was further grown in nutrient broth for another 24 hours. The inoculum equivalent to 10 % (vol) was used for immobilization of the isolates in recycled plastic bottle cups. Biosorption was performed in an aerobic filter bed baffled bioreactor containing the immobilized isolate. This was acclimatized for 1 week, then loading of simulated wastewater containing 1000 ppm zinc. Collections of samples were every four hours for 1 month and analyzed using Atomic Absorption Spectrophotometer.

Chromium biosorption using bacterial isolates

At 25 ppm of Cr, results of digested samples (Table 4.1) showed that three isolates, Acinetobacter sp. junii, Bacillus cereus, and Bacillus toyonesis produced the highest removal rates for chromium (Fig. 4), compared with the lower results with the undigested.

Table 4.1: Effect of sample digestion for the removal rate of Chromium for 4 isolates

Lead biosorption using bacterial isolates

In contrast, the elimination of Pb revealed far better efficiencies for the four bacteria. Four strains attained removal efficiencies in the range of 42 -92% at all concentrations investigated (Figure 5). Bacterial isolate A (Acenitobacter junii) was not effective in removing Pb at higher concentrations of 75 mgL-1 and 100 mg L-1. The highest performance efficiency obtained was 92.43% at 75 mg L-1 that was ascribed to isolate D (Bacillus toyonensis).

Table 5.1: Effect of sample digestion for the removal rate of Lead for 4 isolates.

Nickel biosorption using bacterial isolates

With the case of nickel at 25 ppm the undigested samples (Table 6.1) resulted in the highest removal rate for the three isolates, Acinetobacter sp. tandoii, Bacillus cereus, and Bacillus toyonesis compared with the digested samples, which showed lower removal rate for Ni.

Table 6.1: Effect of sample digestion for the removal rate of Nickel for 4 isolates

Zinc biosorption using bacterial isolates

All the four selected bacterial strains were found to remove zinc at the lower range concentrations of 25mg L-1 and 50 mgL-1 (Figure 7). Between the two levels, greater removal rates were observed at 75 mg-L (digested). Among the bacteria, isolate A ( Acenitobacter junii) and B (Acenitobacter tandoii ) showed removal capability for aqueous media containing 75 mg-L-1 to 100 mgL-1. On the other hand, no observed removal capacity was observed for isolates C (Bacillus cereus) and D (Bacillus toyonensis) at higher concentrations, from 75mg L-1 to 100 mg L-1. Maximum performance efficiency of 89.14% was attained by (Acenitobacter junii) at 75 mgL-1 comparable with Acenitobacter tandoii.

Table 7.1: Effect of sample digestion for the removal rate of zinc for 4 isolates

The observed removal rates of Cu was 38.26% and Zn was 89.14% at a concentration of 75 ppm, Cr was 86.045% at 25 ppm, Ni was 36.42% at 50 ppm and Pb was consistently higher with acid digested waste water samples (using synthetic mine water), which was highest at the rate of 92.43% at 75 ppm.

On Table 1, except for sampling site 1 (Balatoc), the pH values obtained from the other areas were almost neutral and below the DENR standard. The color of the water samples appeared as dark brown, light brown and clear. Turbidity was highest at Balatoc site, a heavily silted and turbulent stream which registered a reading of 621 FAU. All sampling sites exhibited elevated levels of chromium, copper and lead that were way above the regulatory limits (DENR 2016). For other metals however, only site 1 (Balatoc), yielded Zn concentration that was higher than the DENR standard for water quality.

Conclusion

Analysis of untreated water samples collected from the six sites in Itogon, Benguet confirmed the presence of heavy metals at different concentrations. Moreover, all sites contain Cr (VI) with higher than the standard values set by DENR for inland waters. Further, high copper and lead concentrations were also present in some of these sites.

Preliminary screening and isolation carried-out with water samples that were collected from an abandoned mining site yielded high metal tolerant isolates. The selected microbes were molecularly identified as species of bacteria, yeast and fungi. The four identified isolates from Benguet mines contaminated wastewater were capable of removing heavy metals such as: copper, chromium, nickel, zinc and lead.

A total of 150 potential microbial strains were isolated from the wastewater collected from mine tailings in Itogon, Benguet, Mt Province, Philippines after 1 day incubation at room temperature in nutrient agar. After the incubation period, microbial growth was observed in 50 isolates. The fifty isolates were then grown for another 1 day in nutrient agar plate. The 4 promising heavy metals-resistant isolates were then identified at the Philippine Genome Center, UP Diliman, Quezon City. These were identified as isolates A) Acinetobacter sp. junii , B) Acinetobacter sp. tandoii, C) Bacillus cereus and D) Bacillus toyonensis. Experiments showed that the isolate Bacillus toyonensis had the highest capability of removing lead by 92.43% at 75 ppm. Isolate Acinetobacter sp. junii had the percentage removal of zinc by 89.14 % at 75 ppm.

Recommendation

i. Further optimization studies on the parameters for continuous and semi-continuous treatment system for heavy metal contaminated wastewater using the selected isolates.

ii. Further studies on the mechanism of heavy metal removal for the selected isolates in the study.

Acknowledgement

The authors would like to thank National Research Council of the Philippines (NRCP) Department of Science and Technology for funding this project, Benguet Mines in Itogon, Benguet for providing wastewater samples in this study. Department of Science and Technology Cordillera Administrative Region (CAR) for their help in this project. They also acknowledge the technical services of Cynthia Borromeo and Bernard Jude Gutierrez from the Waste Management Section, Environment and Biotechnology Division for the determination of heavy metals content of the samples and for the use of Atomic Absorption Spectrophotometer (AAS).